The cellular machinery involved in mitogenesis is complex, and not fully understood. In general, receptors present on the cell surface bind growth factors, resulting in an activated receptor. In particular, the receptors of interest are endowed with intrinsic tyrosine kinase activity, and are known as tyrosine kinase receptors or TKRs. The activated receptors, in turn, phosphorylate intracellular substrates. These phosphorylated substrates are responsible for a series of events that leads to cell division. This process is generally referred to as "mitogenic signal transduction." The molecular machinery involved in this process is considered to be the "mitogenic signaling pathway."
Growth factors and hormones exert pleiotropic effects on cellular functions, including mitogenic stimulation and modulation of differentiation and metabolism (Ullrich et al., Cell 61:203-212 (1990); Aaronson, Science 254:1146-1153 (1991)). In many cases, these effects are mediated by the interaction of growth factors with cell surface tyrosine kinase receptors (TKRs), resulting in enhanced receptor catalytic activity and tyrosine phosphorylation of intracellular substrates (Ullrich et al., supra; Aaronson, supra). Data regarding the nature of these second messenger systems is still scanty, although some molecules which associate and/or are tyrosine phosphorylated by TKRs have been identified. These include the .gamma. isozyme of phospholipase C (PLC-.gamma.) (Margolis et al., Cell 57:1101-1107 (1989); Meisenhelder et al., Cell 57:1109-1122 (1989); Wahl et al., Mol. Cell. Biol. 9:2934-2943 (1989)); the p21ras GTPase activating protein (GAP) (Molloy et al., Nature 342:711-714 (1989); Kaplan et al., Cell 61:125-133 (1990); Kazlauskas et al., Science 247:1578-1581 (1990)); the raf serine-threonine kinase (Morrison et al., Proc. Natl. Acad. Sci. USA 85:8855-8859 (1988); Morrison et al., Cell 58:649-657 (1989)); the p85 subunit of the phosphatidylinositol 3-kinase (PtdIns-3K); (Coughlin et al., Science 243:1191-1194 (1989); Kazlauskas et al., Cell 58:1121-1133 (1989); Varticovski et al., Nature 342:699-702 (1989); Ruderman et al., Proc. Natl. Acad. Sci. USA 87:1411-1415 (1990); Escobedo et al., Cell 65:75-82 (1991); Skolnik et al., Cell 65:83-90 (1991); Otsu et al., Cell 65:91-104 (1991)) and some cytoplasmic tyrosine kinases (Gould et al., Mol. Cell. Biol. 8:3345-3356 (1988); Kypta et al., Cell 62:481-492 (1990)). These signaling molecules are thought to mediate at least in part the mitogenic effects of TKRs (Ullrich et al., Cell 61:203-212 (1990); Aaronson, Science 254:1146-1153 (1991)).
However, the epidermal growth factor (EGF) receptor (EGFR) does not appear to efficiently interact with known second messenger systems (Fazioli et al., Mol. Cell. Biol. 11:2040-2048 (1991); Segatto et al., Mol. Cell. Biol. 11:3191-3202 (1991)). Thus, there is a need to ascertain the mechanism by which the EGFR functions in mitogenesis, and a particular need to identify and characterize the substrate (if any) of the EGFR.
Errors which occur in the mitogenic signaling pathway, such as alterations in one or more elements of that pathway, are implicated in malignant transformation and cancer. It is believed that in at least some malignancies, interference with abnormal mitogenic signal transduction could cause reversion of cells to a normal phenotype.
In addition, reagents useful in identifying molecular components of the mitogenic signaling pathway would find utility as tumor markers for therapeutic, diagnostic, and prognostic purposes. Furthermore, identification of how such components differ from normal components in malignant tissue might be of significant value in understanding and treating such malignancies. Alterations of the EGFR mitogenic signal transduction pathway have been described in several human tumors. Accordingly, substrates of the EGFR are of particular interest.
Finally, there is a need to identify reagents that can be used to determine the tyrosine kinase activity of particular samples of biological origin. Determination of the tyrosine kinase activity of samples could have value in the therapy, diagnosis, and prognosis of neoplasia and other disorders connected with abnormal mitogenic signaling pathways.
It is therefore an object of the present invention to provide reagents and methods useful in identifying components of the mitogenic signal transduction pathway, for determining tyrosine kinase activity of samples, and for determining how particular components of the pathway in abnormal tissue differ from normal components. In particular, it is an object of the invention to provide reagents and methods that relate to the EGFR substrate(s).